129 B-cells during preclinical phase of rheumatoid arthritis Plasmablast/plasma cells dominate the BCRh repertoire during the pre-clinical phase of RA To determine the phenotype of the (dominant) BCR clones during the at-risk phase, we developed a FACS panel for B cell phenotyping as shown in supplementary figure 3A. As shown in Figure 4A-C, the sequencing results from lysed PBMCs used for the phenotyping of BCR clones are highly comparable to those of lysed peripheral blood cells captured in PAXgene tubes. In 10 RA-risk individuals of the DOMINO study, a total of 23 phenotyping visits were performed. From the peripheral blood collected during these visits, a total of 77 dominant BCRh clones could be detected. FACS sorting allowed us to sequence plasmablast/plasma cell and memory B cell fractions for further downstream sequencing. Of the 77 dominant BCRh clonal signatures in whole blood 88% (n=68) could be retrieved in sequenced data originating from cells with a plasmablast/plasma cell phenotype (Figure 4D-4F and supplementary figure 3C). Fifty percent of these 68 dominant clones could also be retrieved in the memory B cell compartment (Figure 4G-2I). Nine of the 77 dominant BCRh clones detected in peripheral blood could not be found in the plasmablast/plasma cell nor in the memory B cell compartments. To further investigate the behavior and possible origin of these dominant BCRh clones which were predominantly of the plasmablast/plasma cell phenotype, we looked further into their presence over time. Interestingly, 6 and 12 months after the initial phenotyping visit the dominant BCR clones could no longer be found in the plasmablast/plasma cell compartment, but could be traced back as low frequency clones in the memory B cell compartment (Supplementary figure 4A-3D). In summary, plasmablasts/plasma cells carry most of the dominant BCRh signatures, while some BCRh signatures are shared across different B cell phenotypes. 6
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